AMPA receptors (AMPARs) mediate the majority of excitatory glutamatergic synaptic transmission in the central nervous system. Most AMPARs, once bound to glutamate, allow Na+ and K+ flux across the cell membrane, causing neurons to depolarize. However, AMPARs that lack the GluR2 subunit are also permeable to Ca2+. These Ca2-permeable (CP) AMPARs are highly expressed during development when they are essential for activity-dependent plasticity, and this function persists at some synapses throughout adulthood. A biophysical characteristic known as rectification is commonly used to differentiate CP-AMPARs from Ca2+-impermeable (CI) AMPARs. Whereas CP-AMPARs exhibit strong inward rectification, CI-AMPA receptors display linear current-voltage relationships. Inward rectification of CP-AMPARs results from intracellular polyamines that act as open channel blockers to prevent outward current flux. Thus, inward rectification and sensitivity to antagonists that bind at the polyamine site provide biophysical signatures of AMPAR subunit composition and hence Ca2+ permeability, and these characteristics have been widely used to establish rules of AMPAR subunit plasticity. Molecular layer interneurons of the cerebellum provide a well-established model system for understanding AMPAR localization and trafficking because repetitive synaptic stimulation or a single experience of fear triggers a form of plasticity called subunit-switching wherein CP-AMPARs at synapses are replaced by CI-AMPARs from a pool of extrasynaptic AMPARs. Although rectification index and sensitivity to polyamine site toxins are widely used to distinguish between GluR2-containing and -lacking AMPARs, there are many examples from the literature that show these biophysical properties do not exclusively reflect subunit composition. A separate literature has converged on gating models of AMPARs that include multiple conductance states, but the functional implications are unclear. Now, our preliminary data show that CP-AMPAR rectification and pharmacology are sensitive to factors that regulate AMPAR conductance states, potentially complicating the interpretation of results using these biophysical properties as sole proxies of subunit composition. We propose to understand how the multiple sub-conductance states of AMPARs contribute to the hallmark biophysical properties CP-AMPARs. We will use high resolution Ca2+ imaging, heterologous expression systems and genetic manipulation to understand regulation of CP-AMPAR biophysical properties and use that understanding to critically evaluate CP-AMPAR localization and plasticity in cerebellar molecular layer interneurons. AMPAR subunit composition has important functional consequences, ranging from regulating the ability of postsynaptic cells to precisely follow high-frequency synaptic activity and mediating Ca2+ influx that can trigger plasticity or pathology. Successful completion of the proposed studies will reveal novel properties of AMPARs that are essential for understanding their function within synapses and intact circuits in the normal and diseased brain.